G protein-coupled receptors (GPCRs) represent the largest family of signal-transducing molecules known. For example, GPCRs comprise more than 4% of the genes in Caenorhabditis elegans. GPCRs convey signals for light and many extracellular regulatory molecules, such as, hormones, growth factors and neurotransmitters, that regulate every cell in the body. Dysregulation of GPCRs has been found in a growing number of human diseases and GPCRs have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how GPCRs function at the molecular level is an important goal of biological research. We have used receptors for thyrotropin-releasing hormone (TRH) (TRH-Rs) as model GPCRs to study their structure and function. During this year, we have studied several new aspects of TRH-R structure and function. We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical to rat TRH-R2 and 50% identical to mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9 and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of G protein-coupled receptor activity under normal physiologic circumstances. Previous studies showed that rat TRH receptor type 2 exhibits higher basal signaling activity and internalizes more rapidly upon agonist binding than rat TRH receptor type 1. The mouse TRH receptor type 2 (mR2), which we recently cloned, shows a higher basal signaling activity than mR1. Taking advantage of the high degree of sequence homology between mR1 and mR2, we used chimeras/mutants of these receptors to gain insight into the properties of the receptors that influence internalization and basal signaling. Chimeric receptors that have the mR1 extracellular and transmembrane (E/TM) domains with the carboxyl-terminus and intracellular loops of mR2 (R1/R2-tail; R1/R2-I3,tail; R1/R2-I2,3,tail; R1/R2-I1,2,3,tail) exhibited internalization rates and basal activities that were similar to that of mR1. In contrast, a chimeric receptor with the E/TM domains of mR2 and the carboxyl-terminus of mR1 exhibited the more rapid internalization rate and higher basal signaling activity characteristic of mR2. We showed previously that mutation of a highly conserved tryptophan to alanine caused mR1 to exhibit a high basal signaling activity and rapid internalization rate. In contrast, mutation of this tryptophan to alanine in mR2 decreased the rate of internalization and inhibited basal signaling activity. The rates of receptor internalization did not correlate with the binding affinities, coupling efficiencies, or potencies of the receptors. Thus, we observed that receptors with more rapid internalization rates showed relatively higher basal signaling activities whereas receptors with lower basal signaling activities showed slower internalization rates. These data suggest that similar receptor conformations are required for efficient coupling to signaling G proteins and to proteins involved in internalization. Lastly, we synthesized several novel TRH analogs in which the N-terminal polyglutamic acid residue was replaced with polycarboxylic acids and the central histidine was modified with substituted imidazole derivatives and studied their biological properties. These analogs provided new insights into the size of the binding pocket for TRH within TRH-R1.